The sequential introduction into the Americas of chikungunya virus (CHIKV) in 2013 and Zika virus (ZIKV) in 2015 and their co-circulation with dengue virus (DENV) has resulted in an unprecedented public health challenge. The burden of disease caused by these arboviruses (ArboV) is worsened by long-term sequelae in chikungunya and Zika, and by the link between ZIKV infection and severe congenital defects. However, little is known about the clinical presentation of Zika or long-term sequelae in pediatric populations, nor about immune responses to ZIKV infection and possible reciprocal immune enhancement between the two flaviviruses, ZIKV and DENV. Reports of disease enhancement across flavivirus species have been comple- mented by recent reports of in vitro enhancement of ZIKV infection by anti-DENV Abs and vice versa, as both infections generate cross-reactive antibodies, especially in secondary (2) infections. Thus, there is an urgent need to determine whether immune enhancement occurs between DENV and ZIKV and vice versa and to document the clinical outcome. Another consequence of the ArboV co-circulation has been a basic diagnostic challenge. Conventional serological assays are unable to distinguish accurately between 2 DENV and ZIKV infections, impeding regional surveillance systems and studies. Hence, it is critical to develop new serological diagnostic tools that discriminate between anti-ZIKV and anti-DENV antibodies. The proposed study builds on >25 years of collaborative studies with the Nicaraguan Ministry of Health, strengthened by our current, successful IRIDA grant that was initially focused on repeat DENV infections, but expanded to include chikungunya and Zika via administrative supplements as these diseases swept into the region. We aim to address specific and fundamental gaps by leveraging unique, existing research infrastructure and resources to continue two ongoing, complementary studies of pediatric arboviroses in Managua, Nicaragua. The long-running community-based cohort study follows ~3,500 children and allows early capture of Zika, chikungunya, and dengue cases and reconstruction of the immune history of the partici- pants. The hospital study enables the capture and folllow-up of more severe disease cases and has a broader catchment area. In Aim 1, we will characterize the epidemiology of the 3 ArboV, describe the natural history of ZIKV and CHIKV infection including long-term follow-up, and analyze risk factors for clinical outcome of ArboV infections. In Aim 2, we will develop and evaluate traditional and novel next-generation serological diagnostic tools to differentiate ZIKV and DENV infection. In Aim 3, we will examine the effect of prior immunity to DENV/ ZIKV on infection/disease outcome and characterize the quality, breadth and magnitude of immune responses in sequential infections using innovative methods. This proposal addresses major gaps in the knowledge of the natural history and immunology of ZIKV, DENV, and CHIKV infections, and responds directly to a critical need to develop next-generation serological assays to distinguish ZIKV and DENV infections.!